Bottom Line:
Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA.The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology.Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.

ABSTRACTThe small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.

f1: Rapid and reversible GPCR mediated GTP loading of RhoA by p63RhoGEF, measured by a novel high contrast RhoA FRET biosensor.(a) A cartoon of the DORA-RhoA biosensor consisting of full length RhoA (shown in light blue) fused to CFP, connected via a linker to YFP fused to the Rho binding domain of PKN1 (shown in lila) (structures are based on pdb entries 1CXZ, 1MYW and 3ZTF). (b) Average emission spectra (±s.e.m) acquired from living cells (n = 10) of the non-binding RhoA biosensor (RhoAsensor-nb) and the mutant constitutive GTP loaded RhoA biosensor (RhoAsensor-ca). (c) (left) Donor intensity images (top) and phase lifetime images (bottom) of the RhoAsensor-ca (left) and the RhoAsensor-nb (right) with a false-color coded lifetime according to the scale depicted in the combined lifetime histograms of the same experiment (middle). (right) Accumulated FLIM data for RhoAsensor-ca and RhoAsensor-nb, showing the median phase lifetime from multiple cells (at least 8 acquisitions, n = 28 and n = 24, respectively). Box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. (d) Control FRET ratio-imaging experiments in HeLa cells transfected with DORA-RhoA biosensor and only the first 29 a.a. of p63RhoGEF, containing the plasma membrane targeting sequence, show minimal changes in YFP/CFP ratio (n = 29). (e) Time-lapse FRET ratio imaging of HeLa cells transfected with the DORA-RhoA biosensor and RFP-p63RhoGEF (n = 71) show fast reversible increase in YFP/CFP ratio, indicating rapid GTP loading of RhoA upon GPCR stimulation. (f) Average ratio images at three time intervals of a single cell from the experiment shown in (e). (g) Pre-incubation with the Gαq-inhibitor QIC (2 μM) abolishes the DORA-RhoA biosensor response by GPCR stimulation in RFP-p63RhoGEF transfected cells (n = 30). HeLa cells were stimulated with Histamine (100 μM) at t = 40 s and the response was antagonized by the addition of Pyrilamine (10 μM) at t = 160 s. Time traces show the average ratio change of YFP/CFP fluorescence (±s.e.m). Average curves consist of data from at least 3 independent experiments, conducted on different days. Width of the individual images in (f) corresponds to 65 μm.

Mentions:
The kinetics of the guanine exchange reaction of p63RhoGEF on RhoGTPases have so far only been characterized in vitro, using purified proteins16. To investigate the spatial and temporal aspects of RhoA nucleotide binding state in individual living cells, a Dimerization Optimized Reporter for Activation (DORA) RhoA biosensor was employed. The single chain FRET based sensor is based on a previously reported RhoA biosensor21. When RhoA-GDP is converted to RhoA-GTP a PKN1 moiety binds RhoA-GTP, resulting in a high FRET state, which is detected as an increase in sensitized emission over CFP ratio (Fig. 1a). To examine the FRET contrast between the ‘on’ and ‘off’ state of the biosensor, the emission spectra of a non-binding biosensor (RhoAsensor-nb), containing a mutation in PKN1 (L59Q) preventing RhoA binding, and a biosensor containing a constitutively GTP-loaded RhoA mutant (Q63L) (RhoAsensor-ca) were measured in HeLa cells. The average single cell spectra show clearly the CFP emission around 475 nm and the (sensitized) YFP emission around 530 nm (Fig. 1b). The RhoAsensor-ca shows a marked decrease in CFP emission and an increase in YFP emission relative to the RhoAsensor-nb, demonstrating substantial FRET contrast that allows differentiation between the two states. FLIM measurements of the RhoAsensor-ca and RhoAsensor-nb showed that the donor fluorescence lifetime of the constitutively active mutant is reduced compared to that of the non-binding version (median values change from 2.9 to 2.4 ns), demonstrating that the RhoA-GTP state is accompanied by an increase in FRET efficiency (Fig. 1c).

f1: Rapid and reversible GPCR mediated GTP loading of RhoA by p63RhoGEF, measured by a novel high contrast RhoA FRET biosensor.(a) A cartoon of the DORA-RhoA biosensor consisting of full length RhoA (shown in light blue) fused to CFP, connected via a linker to YFP fused to the Rho binding domain of PKN1 (shown in lila) (structures are based on pdb entries 1CXZ, 1MYW and 3ZTF). (b) Average emission spectra (±s.e.m) acquired from living cells (n = 10) of the non-binding RhoA biosensor (RhoAsensor-nb) and the mutant constitutive GTP loaded RhoA biosensor (RhoAsensor-ca). (c) (left) Donor intensity images (top) and phase lifetime images (bottom) of the RhoAsensor-ca (left) and the RhoAsensor-nb (right) with a false-color coded lifetime according to the scale depicted in the combined lifetime histograms of the same experiment (middle). (right) Accumulated FLIM data for RhoAsensor-ca and RhoAsensor-nb, showing the median phase lifetime from multiple cells (at least 8 acquisitions, n = 28 and n = 24, respectively). Box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. (d) Control FRET ratio-imaging experiments in HeLa cells transfected with DORA-RhoA biosensor and only the first 29 a.a. of p63RhoGEF, containing the plasma membrane targeting sequence, show minimal changes in YFP/CFP ratio (n = 29). (e) Time-lapse FRET ratio imaging of HeLa cells transfected with the DORA-RhoA biosensor and RFP-p63RhoGEF (n = 71) show fast reversible increase in YFP/CFP ratio, indicating rapid GTP loading of RhoA upon GPCR stimulation. (f) Average ratio images at three time intervals of a single cell from the experiment shown in (e). (g) Pre-incubation with the Gαq-inhibitor QIC (2 μM) abolishes the DORA-RhoA biosensor response by GPCR stimulation in RFP-p63RhoGEF transfected cells (n = 30). HeLa cells were stimulated with Histamine (100 μM) at t = 40 s and the response was antagonized by the addition of Pyrilamine (10 μM) at t = 160 s. Time traces show the average ratio change of YFP/CFP fluorescence (±s.e.m). Average curves consist of data from at least 3 independent experiments, conducted on different days. Width of the individual images in (f) corresponds to 65 μm.

Mentions:
The kinetics of the guanine exchange reaction of p63RhoGEF on RhoGTPases have so far only been characterized in vitro, using purified proteins16. To investigate the spatial and temporal aspects of RhoA nucleotide binding state in individual living cells, a Dimerization Optimized Reporter for Activation (DORA) RhoA biosensor was employed. The single chain FRET based sensor is based on a previously reported RhoA biosensor21. When RhoA-GDP is converted to RhoA-GTP a PKN1 moiety binds RhoA-GTP, resulting in a high FRET state, which is detected as an increase in sensitized emission over CFP ratio (Fig. 1a). To examine the FRET contrast between the ‘on’ and ‘off’ state of the biosensor, the emission spectra of a non-binding biosensor (RhoAsensor-nb), containing a mutation in PKN1 (L59Q) preventing RhoA binding, and a biosensor containing a constitutively GTP-loaded RhoA mutant (Q63L) (RhoAsensor-ca) were measured in HeLa cells. The average single cell spectra show clearly the CFP emission around 475 nm and the (sensitized) YFP emission around 530 nm (Fig. 1b). The RhoAsensor-ca shows a marked decrease in CFP emission and an increase in YFP emission relative to the RhoAsensor-nb, demonstrating substantial FRET contrast that allows differentiation between the two states. FLIM measurements of the RhoAsensor-ca and RhoAsensor-nb showed that the donor fluorescence lifetime of the constitutively active mutant is reduced compared to that of the non-binding version (median values change from 2.9 to 2.4 ns), demonstrating that the RhoA-GTP state is accompanied by an increase in FRET efficiency (Fig. 1c).

Bottom Line:
Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA.The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology.Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.

ABSTRACTThe small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.